PROJECT 2 SUMMARY The overall objective of the Project 2 is to utilize a newly discovered, active transendothelial transport pathway, the caveolae pumping system, in order to provide an effective solution to the delivery and toxicity problem of chemotherapeutics in metastatic breast cancer treatment. In order to address these problems and significantly improve therapeutic outcome we propose to develop novel antibody-drug conjugates (ADCs) that exploit our newly discovered endothelial cell (EC) caveolae targeting system in order to sidestep passive delivery problem of modern chemotherapeutics. We have established that EC caveolae in preclinical models can rapidly and specifically pump anti-Annexin A1 antibodies and attached cargo across the vascular endothelial barrier directly into solid tumors. Based on this discovery, we propose to design novel EC caveolae-targeted ADCs for greatly enhanced efficacy in tumor destruction in breast cancer. Our main hypothesis is that immunoconjugates that fully utilize the advantages of the EC caveolae-pumping system will dramatically increase delivery of chemotherapeutic drugs into tumors, thereby requiring much lower dosages and dramatically enhancing efficacy of treatment. This hypothesis will be tested by the following specific aims: In Aim 1, we will design and synthesize Pt(II)-carboxymethyl dextran (CMD) ADCs and optimize carboxylate derivatization of CMD, conjugation chemistry, and Pt(II) loading in order to maximize binding affinity toward human Annexin A1 protein. We will test and optimize physicochemical properties, stability on storage, in human plasma, and Pt(II) release kinetics in the tumor interstitium. In Aim 2 we will assess therapeutic efficacy of the ADCs in metastatic cancer models. The efficacy of our targeted delivery system will be examined in MDA-231-LM2-4, and rat tumor models with 13762 breast cancer cells metastasized to lung using whole-body animal imaging with X-ray/CT. In Aim 3 we will examine the therapeutic efficacy of the ADCs in tumors with human blood vessels using intravital microscopy (IVM). We will monitor tumor size in response to therapy in human-on-human IVM model using BT-474 tumor spheroids, and in patient-derived xenografts (PDX) IVM model. In this Aim we will also translate our select preclinical candidate for clinical testing, where successful targeted therapeutic will be selected as clinical candidates for cGMP production (Core B) for subsequent testing in a Phase 1 safety trial in Project 3. Antibodies for conjugation to form ADCs will be provided by Core B. Core B will also provide quality control analysis of ADCs and confirmation of antibody binding affinity following conjugation. Tumor targeting, delivery and accumulation of these novel therapies will be assessed in rodents using multi-modality imaging services provided by Core C, which includes X-ray/CT, SPECT-CT, IVM, and histology. Core E will handle the biostatistical analysis of data generated during the course of the project. The long-term goal is to translate our key basic discoveries into unique, innovative delivery platform in order to improve therapeutic efficacy of human cancer therapy.